Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Abstract

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

Reaction Initiation in Enzyme Crystals by Diffusion of Substrate

“…With serial micro-crystallography, the initiation of a reaction by diffusion is easily conceivable since this method automatically provides crystals that are three dimensionally thin, fulfilling the conditions envisioned by the early protein crystallographers. To make reaction initiation by diffusion feasible, a number of approaches are currently pursued: the mix-and-inject technique using a GDVN [8,12,[39][40][41], acoustic ligand droplet ejection onto a chip [42] or a moving tape-containing crystals [43], and the 'fountain pen' method that also uses a tape drive [44] to transport the microcrystals towards the X-rays. All these techniques mix the ligand with the crystals and they do so reliably and rapidly.…”

“…Fifty years after the early protein crystallographers envisioned such an approach, it became reality, and can now be used to investigate all sorts of enzymatic reactions. MISC seems to be established now as an accepted technique to investigate enzymatic reactions [12,39,41,42]. The crystals (green) are mixed with the substrate (Ceftriaxone, yellow).…”

Advances in methods for atomic resolution macromolecular structure determination

“…The long-sought mechanism of photosynthetic water splitting by photosystem II has been revealed using ultrafast time-resolved crystallography paired with simultaneous X-ray spectroscopy 223 – 225 . Covalently bound enzyme–substrate reaction intermediates, which had been theorized but never observed, have been identified 226 , 227 . Additionally, a substantial effort has been made to expand time-resolved measurements to systems that do not have any photoactivity, making these experiments amenable to essentially any protein of interest.…”

“…Additionally, a substantial effort has been made to expand time-resolved measurements to systems that do not have any photoactivity, making these experiments amenable to essentially any protein of interest. New perturbation methods that have been successfully utilized include temperature-jump 228 , rapid mixing 226 , 227 , ligand or substrate photocaging 229 , and rapid application of electric fields 230 . Finally, it is worth noting that the success of time-resolved crystallography has inspired the more recent development of time-resolved cryo-EM 231 .…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…Use of caged substrates [122] could bypass the need for rapid diffusion by bringing the substrate at close vicinity, but this requires sophisticated chemistry and may not be applicable to many ligands. TR-SFX experiments using diffusion of a substrate as a pump trigger is usually based on a "mix-and-inject" approach [193][194][195][196][197][198], in which a substrate is rapidly mixed into crystal suspension to initiate a reaction before delivery into XFEL pulses after a time delay. This requires a different jet set up for sample delivery.…”